No description available.

The objective of this project was to explore the role of nutrients, mainly glucose and fatty acids, in the regulation of insulin gene expression via the complex interaction factors and positive or negative <i>cis</i>-acting DNA elements located throughout the promoter region, which is positioned up to 300bp. Insulin is a vital endocrine hormone, for the regulation of growth and metabolism. Synthesis of insulin occurs in pancreatic cells. The regulation of tissue- and temporal-specific insulin gene expression and its activation in response to extracellular inducers are two fundamental processes that attract many molecular biologists. Therefore to achieve the objective three major approaches were undertaken in this project. 1. Characterizing and identifying the transcription factors that bound to the negative regulatory element (NRE) within the region -258 to -280 of the human insulin promoter. This was done by applying a promising and unique method including a proteomic approach. The major findings propose NRE bind multiple positive and negative factors. 2. Studying in detail the effect of glucose on the factors that bound to the - 230 to -280 region which included NRE binding factors and PDX-1 which binds A-boxes, in various pancreatic rodent cell lines and isolated human islet of Langerhans. This study shows that the glucose stimulating response of the NRE in b cell lines and human islets does not require synergy between adjacent sequence elements such as E2 binding USF and A5 binding the PDX-1. Therefore, the NRE binding factors are able to demonstrate convincingly the existence of short-term transcriptional control of the insulin gene regulated by glucose. 3. Studying the expression of the lipogenic transcription factor SREBPs in the islets of Langerhans and their role in controlling the expression of several genes. The present study provided the first demonstration of the role of SREBPs in mediating the effect of nutrient signals, particularly glucose, on the insulin promoter and its mRNA.

The translation of a series of <I>cat </I>mRNAs containing either the <I>HSP26 </I>5'- leader or various artificial 5'-leaders (Vega Laso <I>et al., </I>1993) were analysed during growth. From this study, the relative translational efficiencies of these mRNAs were shown to vary from 2% to 100% during mid-exponential phase as observed previously (Vega Laso <I>et al.,</I>1993). However, upon analysing the translation of the various <I>cat </I>constructs during growth, their relative translational efficiencies did not change significantly as yeast cells approached stationary phase. A new set of <I>lacZ </I>mRNAs carrying different natural 5'-leaders (<I>PGK1, PYK1, RpL3, Rp29, GDH1, HSP26, HSP12 </I>and <I>TH14</I>) were constructed. These <I>lacZ </I>mRNAs were placed under the control of the promoters taken from genes expressed during different phases of growth (<I>PGK1 </I>and <I>HSP26</I>). Even though the various <I>PGK1-lacZ </I>and <I>HSP26-lacZ </I>mRNAs were translated differentially, the ability of these mRNAs to compete for the translational apparatus did not appear to change as cells entered stationary phase. The translation of a variety of natural mRNAs encoding a wide range of functions was then analysed by determining their polysomal distribution at various points during growth. Irrespective of the growth phase, a large proportion of each mRNA was detected in the polysomal fractions, suggesting that they continued to be translated in stationary phase. Overall, the data strongly suggest that, under the conditions tested, an excess translational capacity exists in stationary phase yeast cells. Hence gene expression may be largely regulated by transcription upon entry to stationary phase.

No description available.

No description available.

Cancer is a devastating disease for human society with thousands of deaths and estimated new cases every year around the globe. Intensive research efforts on understanding the disease progression and determining effective diagnostics and therapeutics have been employed for over one hundred years. Throughout this time, and in particular during the last two decades, computational-based methods have gained increasing importance in cancer biology research by providing significant advantages in the analysis and interpretation of high-throughput data at the molecular and genomic levels. More specifically, after completion of the Human Genome Project in 2003, and with the Cancer Human Genome Project underway, high-throughput biological assays (e.g., microarray chips, next generation sequencing machines) have supplied researchers thousands of measurements per experimental sample. The massive amount of related data has oftentimes been challenging to interpret and translate, particularly in cancer biology and therapeutics. This thesis reports the results of three independent studies in which high-throughput gene expression is computationally analyzed to address longstanding issues in cancer biology. Two of the studies utilize data from ovarian cancer patients while the third involves data collected from pancreatic cancer patients. In Chapter 1, I address the importance of personalized profiling in pancreatic cancer ; in Chapter 2 the role of cancer stroma in the progression of ovarian cancer and in Chapter 3 evidence for the role of epithelial-to-mesenchymal transition (EMT) in ovarian cancer metastasis. More specifically, Chapter 1 emphasizes the power of personalized molecular profiling in unmasking unique gene expression signatures that correspond to each individual patient. These individual expression patterns (individual profiling), which may be overlooked by the traditional methods of gene signatures enriched in groups of afflicted individuals (group profiling), can provide valuable information for more successful targeted therapies. In order to address this issue in pancreatic cancer, comparisons of the most significantly differentially expressed genes and functional pathways were performed between cancer and control patient samples as determined by group vs. personalized analyses. There was little to no overlap between genes/pathways identified by group analyses relative to those identified by personalized analyses. These results indicated that personalized and not group molecular profiling is the most appropriate approach for the identification of putative candidates for targeted gene therapy of pancreatic and perhaps other cancers with heterogeneous molecular etiology. Chapter 2, also with strong implications on personalized molecular profiling, unveils the functional variability of the tumor microenvironment among ovarian cancer patients. The purpose of this study was to investigate the process of microenvironmental stroma activation in human ovarian cancer by molecular analysis of matched sets of cancer and surrounding stroma tissues from individual patients. Expression patterns of genes encoding signaling molecules and compatible receptors in the cancer stroma and cancer epithelia samples indicated the existence of two sub-groups of cancer stroma with different propensities to support tumor growth. These results demonstrated that functionally significant variability exists among ovarian cancer patients in the ability of the microenvironment to modulate cancer development. Chapter 3 aims to uncover the molecular mechanisms that underlie the metastatic process with the hope that such knowledge may lead to more effective therapeutic treatments. For this purpose, pathological and molecular analyses were conducted in 14 matched sets of primary and metastatic samples from late staged ovarian cancer patients. Pathological examination revealed no morphological differences between any of the primary and metastatic samples. In contrast, gene expression analyses identified two distinct groups of patient samples. One group displayed essentially identical expression patterns to primary samples isolated from the same patients. The second group displayed expression patterns significantly different from primary samples isolated from the same patients. Predominant among the differentially expressed genes characterizing this second class of metastatic samples were genes previously associated with epithelial-to-mesenchymal transtion (EMT). These results supported a role of EMT in at least some ovarian cancer metastases and demonstrated that indistinguishable morphologies between primary and metastatic cancer samples is not sufficient evidence to negate the role of EMT in the metastatic process. / The data related to the ovarian cancer work discussed in this dissertation are available at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38666

Differentiation and gene expression in the oomycete Achlya ambisexualis ( strain E87) were studied during three responses, sexual antheridium formation, asexual sporangium formation and heat shock. Polyacrylamide gel electrophoresis of proteins labelled in vivo and in cell free systems directed by poly(A) mRNA showed that during formation of antheridia only one change was detectable in the proteins synthesized. No detectable changes were observable in the translatable poly(A) RNA population indicating that post-translational events may be required for differentiation. During early sporangium formation several proteins showed changes in relative rates of synthesis. These correlate with similar changes in cell free translation products suggesting a transcriptional level of regulation. Nucleic acid hybridization analyses of mRNA populations indicated that many new sequences appear late and accumulate in the spore. These late transcripts are probably utilized during spore germination. During heat shock several proteins showed rapid increases in rates of synthesis; these seem to be controlled at the level of transcription.

Transport, a common cause of stress in livestock, has been documented to increase cortisol, and epinephrine in goats. However, little is known about the timing of changes in the immune system in these stressed animals. The objective of this study was to determine whether expression of immune-related genes changes in goats that are exposed to transport stress. In this study, 15 Spanish-Boer goats ranging from 3 to 4 yrs of age were transported for 12 h. Goats were divided into 5 groups of 3 and placed in 1.219 m x 1.219 m pens. Blood samples were collected via jugular veni-puncture from each animal at 0 h, 3 h, 6 h, 9 h, and 12 h of transport, plasma and leukocytes were harvested for cortisol analysis and PCR analysis for gene expression. Data was analyzed using trailer location (group) as the experimental unit in a mixed model, repeated measures analysis of variance with compound symmetry and autoregressive covariance structures, depending on the best fit for each model. Percent weight losses were analyzed using a diagonal covariance mixed model. Hourly temperature humidity index (THI) values inside the trailer and from the shade were analyzed using a two-independent sample T-test. Cortisol concentrations were significantly elevated during transport (P<.049), indicating that goats experienced stressful events during hours of transport. Cortisol concentrations peaked after 6 hours, and returned to near basal concentrations after 12 h of transport. There was an overall trend for greater expression of many of the genes of interest to increase expression after 12 h of transport, but none were significantly different from pre-transport expression values. Overall, the data suggests that the goats transported during this study experienced transport stress, as indicated by the elevation in cortisol concentrations, but did not have significant changes in expression of the immune-related genes after 12 h of transport.

The PsbL, PsbM and PsbT subunits of photosystem II (PSII) are single-helix membrane-spanning proteins found at the monomer-monomer interface that may stabilize the dimeric complex. This study has characterised strains of Synechocystis sp. PCC 6803 where psbL, psbM and psbT have been interrupted by the insertion of antibiotic-resistance cassettes. The [Delta]PsbL strain exhibited slowed growth that correlated with a disruption in PSII assembly leading to an accumulation of CP43-less PSII monomers. Moreover, the [Delta]PsbL:[Delta]PsbM and [Delta]PsbL:[Delta]PsbT double mutants were not photoautotrophic. In contrast, the [Delta]PsbM and [Delta]PsbT strains grew photoautotrophically and supported oxygen evolution, albeit at reduced rates compared to wild type. S-state analyses showed that the removal of PsbM or PsbT did not affect the donor side reactions of PSII, which includes the oxidation of water, however, the removal of PsbT impaired electron flow between Q[A] and Q[B] on the acceptor side of PSII. Blue-Native PAGE revealed that removal of either PsbM or PsbT was insufficient to entirely disrupt dimer formation; however, the combined removal of PsbM and PsbT resulted in the predominance of monomeric forms of PSII in the [Delta]PsbM:[Delta]PsbT strain. Under high light (2 mE m⁻� s⁻� at 30�C), [Delta]PsbM and [Delta]PsbT cells were considerably more susceptible to photoinactivation than wild type; however, they were able to fully recover in a protein synthesis-dependent manner when returned to moderate light levels (0.03 mE m⁻� s⁻�). A requirement for Psb27 was found in the protein-synthesis-dependent recovery of photoinactivated [Delta]PsbT cells. More significantly, an absolute functional requirement was found for Psb27 in the [Delta]PsbM strain, where functional PSII complexes are not assembled in the absence of Psb27. These results suggest that Psb27 is critical for PSII assembly in the absence of PsbM, and also for the protein-synthesis-dependent recovery of PSII in the absence of PsbT. Moreover, in addition to Psb27, the PsbU subunit of the oxygen-evolving complex was also found to be an absolute functional requirement in the [Delta]PsbM strain, where functional PSII centres are not assembled when both PsbM and PsbU are absent. It appears, therefore, that PsbM has crucial functional interactions with specific extrinsic proteins located in the vicinity of the oxygen-evolving complex. Interestingly, the [Delta]PsbM strain was also found to have a high susceptibility to suppressor mutations, indicating it has important functional roles in the cyanobacterial cell.

Polyreactive immunoglobulins (Ig) exhibit a capacity to recognise multiple, structurally dissimilar antigens through a single combining site. This characteristic differentiates these Igs from monoreactive Igs which bind to a single antigen, usually with high specificity and affinity. Chronic B lymphocytic leukaemia (B CLL) is a malignancy identified by the incessant accumulation, in the peripheral circulation, of B lymphocytes of a mature and resting morphology. B CLL malignant cells generally express both surface IgM and the pan T cell antigen CD5. Moreover, the IgM on the surface of these CD5 positive B CLL cells is frequently polyreactive. This thesis examines the structural diversity found in the combining sites of B CLL derived Igs in an attempt to elucidate the structural basis of polyreactive antigen binding displayed by a significant proportion of human Igs. The genes encoding the variable (V) domains of five B CLL derived IgM antibodies (Bel, Tre, Yar, Hod and Jak) were cloned and sequenced (Chapter Two). When the light chain V domain genes were aligned with the closest germline VL and JL coding DNA sequences it was determined that there was either a complete absence of somatic mutation (Tre, Yar and Jak) or a minimal number of mutations (Bel and Hod) present in the rearranged VL domain genes. A remarkable fidelity in the splicing of VL to JL genes was noted suggesting that the diversity, normally introduced through variability of splicing VL to JL, is reduced in Igs expressed by B CLL cells. Furthermore, the markedly reduced primary structural diversity was highlighted when two of the VL domain genes (Yar and Hod) were found to be different in sequence by only four nucleotides and two amino acids. The heavy chain V domain genes of the same five Igs were sequenced in another study (Brock, 1995), however, it was interesting to analyse the sequences of the VH domain genes and compare them with the VL domain genes. The naive or gerrnline nature of the B CLL antibodies was reflected in the VH genes by either an absence or a low frequency of mutations within these sequences compared with germline immunoglobulin gene sequences. No obvious conserved motif, which could be related to polyreactivity, was observed when the primary protein sequence was analysed for distribution of identical or similar amino acids. Thus, homology modelling was used to construct three-dimensional models of the Fv (VL-VH) portions of the five B CLL IgM molecules to examine the structures of the combining sites of these Igs (Chapter Three). Framework regions were constructed using X-ray coordinates taken from highly hon~ologous human variable domain structures. Complementarity determining regions (CDR) were predicted by grafting loops, taken from known Ig structures, onto the Fv framework models. The CDR templates were selected, where possible, to be of the same length and of high residue identity or similarity. If a single template CDR was not appropriate to model a particular CDR the loop was built from loop sterns of known conformation, followed by chain closure with a p-turn. Template models were refined using standard molecular mechanics simulations. The binding sites were either relatively flat or contained a deep cavity at the VL-VH domain interface. Further differences in topology were the result of some CDR loops protruding into the solvent. Examination of the electrostatic molecular surface did not reveal a common structural feature within the binding sites of the five polyreactive Fv. While two of the binding cavities were positively charged the other three structures displayed either negatively charged or predominantly hydrophobic combining sites. These findings suggested that a diversity of structural mechanisms are involved in polyreactive antigen binding. Rcsidues within CDRs which have aromatic side-chains and are partially exposed to solvent were distributed across large regions of the combining sites. It is possible that these aromatic residues are responsible for the conserved binding to mouse Igs observed (Chapter Two) for the B CLL derived polyreactive IgM molecules. Two Fv molecules (Be1 and Tre) were cloned as dicistronic constructs, into the bacterial expression vector pFLAG. The expression of the Fvs was fully characterised and unfortunately the VL and VH of Be1 and Tre Igs did not associate in an appropriate manner to yield large quantities of purified Fv (Chapter Four). Expression of correctly folded and stabilised fragments of human polyreactive immunoglobulins would enable the structural basis for the polyreactive binding phenomenon to be fully explored using protein crystallography.